Diabetes is a common metabolic disease frequently associated with defects in insulin signaling. The model organism C. elegans has emerged as an excellent system to study the insulin signaling pathway, which is highly conserved between humans and C. elegans. This proposal seeks to utilize mass spectrometry-based protein identification and quantitation technologies developed in our lab to identify and characterize protein complexes and novel components of the C. elegans insulin signaling pathway. DAF-2 (C. elegans insulin receptor), AGE-1 (PI 3-kinase), and AKT-1 (AKT kinase) complexes labeled with a tandem affinity purification tag will be purified from C. elegans and analyzed by mass spectrometry. Downstream targets of DAF-16 (C. elegans forkhead transcription factor) will be identified by quantitative proteomic analysis of wild type and daf-16 mutants labeled by 14N and 15N. Novel insulin signaling components identified in this study will be inactivated using RNAi and C. elegans insulin signaling assays will be used to assess their physiological significance. Proteomic analysis of the insulin signaling pathway in conjunction with C. elegans genetics will lead to a better understanding of insulin signaling and provide new therapeutic targets to battle diabetes.